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Appl Environ Microbiol, June 1998, p. 1997-2005, Vol. 64, No. 6
Departments of Molecular
Biology1 and
Animal
Science2, University of Wyoming, Laramie,
Wyoming 82071
Received 9 January 1998/Accepted 24 March 1998
A collection of pediocin AcH amino acid substitution mutants was
generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an
Escherichia coli malE plasmid that directs the secretion of
maltose binding protein-pediocin AcH chimeric proteins and by screening
transformant colonies for bactericidal activity against
Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol.
64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S,
C14Y, G37E, G37R, and C44W) were completely inactive against the
pediocin AcH-sensitive strains L. plantarum NCDO955,
Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc
mesenteroides Ly. A C24S substitution mutant constructed by other
means also was inactive against these bacteria. Nine other mutants
(K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to ~60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed ~2.8-fold-higher
activity against this indicator. About one half of the mutations mapped
to amino acids that are conserved in the pediocin-like family of
bacteriocins. All four cysteines were found to be required for
activity, although only C9 and C14 are conserved among pediocin-like
bacteriocins. Several basic amino acids as well as nonpolar amino acids
located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Isolation and Characterization of Pediocin AcH
Chimeric Protein Mutants with Altered Bactericidal Activity
and
*
Corresponding author. Mailing address for Kurt W. Miller: Department of Molecular Biology, P.O. Box 3944, University of
Wyoming, Laramie, WY 82071-3944. Phone: (307) 766-2037. Fax: (307)
766-5098. E-mail: kwmiller{at}uwyo.edu. Mailing address for Bibek
Ray: Department of Animal Science, P.O. Box 3684, University of
Wyoming, Laramie, WY 82071-3684. Phone: (307) 766-3140. Fax: (307)
766-2350. E-mail: labcin{at}uwyo.edu.
Present address: Middle East Technical University, Ankara,
Turkey.
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